The Mentored Clinical Development Award (MCSDA) will provided the candidate, Dr. Darren Machule an opportunity to develop into an independent researcher that will provide both translational and basic science research in the field of tooth development and biomineralization. This award will allow the candidate to finish his Ph.D. training in Oral Biology and complete a specialty program in Endodontics at University of California San Francisco (UCSF). The Oral Biology program at UCSF provides a unique environment that has proven to be successful in educating students that pursue productive academic careers. This environment correlates well with the candidate's long-term goals of devotion to academia. The candidate is interested in running his own laboratory in the future coupled with teaching and mentoring undergraduate and graduate students. The foundation for the candidate's research career development plan relies on building a base of knowledge and expertise in the tooth development field. The MCSDA award will facilitate the candidate's goals. This proposal is based on the premise that the enamel protein ameloblastin plays a role in tooth development and biomineralization. The function of ameloblastin is still unknown, yet it is the second most common protein in the developing tooth enamel matrix. Not only is ameloblastin unique in its tissue specificity to ameloblasts, odontoblasts, and cementoblasts, but it remains highly conserved through out several species. This conservation includes three potential phosphorylation sites, with one of ameloblastin's two splice variants adding a key tyrosine into the correct position for a tyrosine kinase phosphorylation site. The different phosphorylation pattern of ameloblastin's two splice variants may lead to different functions for this protein. The conservation of this protein and its tissue specificity builds a strong argument for a predominant role in tooth development. I propose that the ameloblastin protein is phosphorylated, and further that ameloblastin is essential for enamel development with the splice variants of this protein providing distinct roles. The specific aims are as follows: 1) to determine the spatial and temporal pattern of ameloblastin splice variant expression during tooth development, 2) to test the hypothesis that the tyrosine residue at the C-terminus of the predicted tyrosine kinase phosphorylation site provides a tyrosine-phosphorylation site in vivo, and 3) to gain increased understanding of ameloblastin function during tooth development using a tooth organ culture system and function-perturbing antibodies. By understanding the roles of the tooth enamel protein ameloblastin we will advance our understanding of biomineralization and be closer to engineered tooth development.